All Research Areas

Nonlinear microscopy

Nonlinear optical microscopy is generally divided into two categories: incoherent or coherent. Incoherent microscopy produces an optical signal whose phase is random and whose power is proportional to the concentration of radiating molecules. Fluorescence is a common example of an incoherent signal. Nonlinear versions of fluorescence microscopes are based on the simultaneous absorption of two or more photons, the most well known being two-photon excited fluorescence (TPEF) microscopy. In TPEF microscopy, two excitation photons from a pulsed laser (typically a Ti:sapphire laser of wavelength 700nm-1000nm) combine to excite a fluorescent molecule. The molecule then releases its excitation energy as a fluorescence photon (typically a visible wavelength). Because the excitation is nonlinear, the fluorescence is confined to the focal center of the laser beam, and fluorescence power decays as 1/z2, where z is the axial distance away from the focus (see figure). TPEF microscopy therefore confers 3D-imaging with out-of-focus background rejection similar to a confocal microscope. The advantage of TPEF microscopy over confocal microscopy is that it can penetrate deeper in thick tissue.

Coherent microscopes produce optical signals whose phase is rigorously prescribed by a variety of factors including the excitation light phase and the geometric distribution of the radiating molecules. Coherent signal power is proportional to the concentration of radiating molecules squared. Nonlinear versions of coherent microscopy are based on the simultaneous scattering of two or more photons. Examples are second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. SHG signals are highly sensitive to molecular orientation.

We are currently working on further developments of TPEF microscopy and its applications to brain imaging. In many cases, we combine TPEF microscopy with simultaneous SHG microscopy or autoconfocal microscopy.

Publications Related to this Research Area

Pyramidal cells accumulate chloride at seizure onset

K. P. Lillis, M. A. Kramer, J. Mertz, K. J. Staley and J. A. White,

Neurobiology of Disease

Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity.

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Control of local intracellular calcium concentration with dynamic-clamp controlled 2-photon uncaging

E. Idoux and J. Mertz,


The variations of the intracellular concentration of calcium ion ([Ca2+]i) are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca2+]i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca2+]i changes is difficult to attain. The tools designed so far to modify [Ca2+]i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electrooptical technique to quantitatively set [Ca2+]i, in real time and with sub-cellular resolution, using two-photon Ca2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca2+]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca2+ channels, and the standardization of [Ca2+]i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.

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Practical implementation of log-scale active illumination microscopy

K. K. Chu, D. Lim and J. Mertz,

Biomedical Optics Express

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.

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Two-photon fluorescence microscopy with differential aberration imaging

K. K. Chu, A. Leray, T. G. Bifano and J. Mertz,

Proceedings of the SPIE

We report our progress in the development of Differential Aberration Imaging (DAI), a technique that enhances two-photon fluorescence (TPEF) microscopy by improving rejection of out-of-focus background by means of a deformable mirror (DM). The DM is used to intentionally add aberrations to the imaging system, which causes dramatic losses to in-focus signal while preserving the bulk of the out-of-focus background. By taking the difference between TPEF images with and without added aberrations, the out-of-focus portion of the signal is further rejected. We now introduce an implementation of DAI using a new type of DM that can be produced at much lower cost.

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Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

K. P. Lillis, A. Eng, J. A. White and J. Mertz,

Journal of Neuroscience Methods

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5 mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

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Bonding of macromolecular hydrogels using perturbants

G. M. Price, K. K. Chu, J. G. Truslow, M. D. Tang-Schomer, A. P. Golden, J. Mertz and J. Tien,

Journal of the American Chemical Society

This work describes a method to bond patterned macromolecular gels into monolithic structures using perturbants. Bonding strengths for a variety of solutes follow a Hofmeister ordering; this result and optical measurements indicate that bonding occurs by reversible perturbation of contacting gels. The resulting microfluidic gels are mechanically robust and can serve as scaffolds for cell culture.

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Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy

A. Leray, K. Lillis and J. Mertz,

Biophysical Journal

When a two-photon excited fluorescence (TPEF) microscope is used to image deep inside tissue, out-of-focus background can arise from both ballistic and nonballistic excitation. We propose a solution to largely reject TPEF background in thick tissue. Our technique is based on differential-aberration imaging with a deformable mirror. By introducing extraneous aberrations in the excitation beam path, we preferentially quench in-focus TPEF signal while leaving out-of-focus TPEF background largely unchanged. A simple subtraction of an aberrated, from an unaberrated, TPEF image then removes background while preserving signal. Our differential aberration (DA) technique is simple, robust, and can readily be implemented with standard TPEF microscopes with essentially no loss in temporal resolution when using a line-by-line DA protocol. We analyze the performance of various induced aberration patterns, and demonstrate the effectiveness of DA-TPEF by imaging GFP-labeled sensory neurons in a mouse olfactory bulb and CA1 pyramidal cells in a hippocampus slice.

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Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination

K. K. Chu, D. Lim and J. Mertz,

Optics Letters

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

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Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging

A. Leray and J. Mertz,

Optics Express

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. An added benefit of our technique is that it leads to somewhat improved image resolution.

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Membrane potential detection with second-harmonic generation and two-photon excited fluorescence: a theoretical comparison

T. Pons and J. Mertz,

Optics Communications

We theoretically compare the performance of TPEF and SHG microscopy for membrane potential imaging. We argue that electrochromic TPEF and SHG membrane potential responses are reflections of the same phenomenon, and can be described in a unified manner as resulting from the linear Stark effect. We also show that TPEF and SHG exhibit similar sensitivities in the case of both electrochromic and orientational response mechanisms. Despite their similar sensitivities, SHG nevertheless presents advantages over TPEF for membrane potential imaging because of its remarkable spatial and spectral contrast, and because of its insensitivity to non-radiative excited-state damping mechanisms.

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Effects of (multi)branching of dipolar chromophores on photophysical properties and two-photon absorption

C. Katan, F. Terenziani, O. Mongin, M. H. V. Werts, L. Porrès, T. Pons, J. Mertz, S. Tretiak and M. Blanchard-Desce,

Journal of Physical Chemistry A

To investigate the effect of branching on linear and nonlinear optical properties, a specific series of chromophores, epitome of (multi)branched dipoles, has been thoroughly explored by a combined theoretical and experimental approach. Excited-state structure calculations based on quantum-chemical techniques (time-dependent density functional theory) as well as a Frenkel exciton model nicely complement experimental photoluminescence and one- and two-photon absorption findings and contribute to their interpretation. This allowed us to get a deep insight into the nature of fundamental excited-state dynamics and the nonlinear optical (NLO) response involved. Both experiment and theory reveal that a multidimensional intramolecular charge transfer takes place from the donating moiety to the periphery of the branched molecules upon excitation, while fluorescence stems from an excited state localized on one of the dipolar branches. Branching is also observed to lead to cooperative enhancement of two-photon absorption (TPA) while maintaining high fluorescence quantum yield, thanks to localization of the emitting state. The comparison between results obtained in the Frenkel exciton scheme and ab initio results suggests the coherent coupling between branches as one of the possible mechanisms for the observed enhancement. New strategies for the rational design of NLO molecular assemblies are thus inferred on the basis of the acquired insights.

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Two-photon absorption and fluorescence with quadrupolar and branched chromophores – effect of structure and branching

L. Porrès, O. Mongin, C. Katan, M. Charlot, B. K. G. Batthula, V. Jouikov, T. Pons, J. Mertz and M. Blanchard-Desce,

Journal of Nonlinear Optical Physics & Materials

The photophysical and two-photon absorption (TPA) properties of three homologous quadrupolar and one related three-branched chromophores were investigated. Design of the quadrupoles is based on the symmetrical functionalization of a biphenyl core. Modulation of the nonlinear absorptivity/transparency/photostability trade-off can be achieved by playing with the twist angle of the core and on the spacers (phenylene-vinylene versus phenylene-ethynylene). The quadrupolar chromophores combine high TPA cross-sections, high fluorescence quantum yield and solvent sensitive photoluminescence properties. The branched structure exhibits spectrally broadened TPA in the NIR region (up to 3660 GM at 740 nm measured in the femtosecond regime) but reduced sensitivity to the environment.

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Two-photon absorption and fluorescence in nanoscale multipolar chromophores: effect of dimensionality and charge-symmetry

L. Porrès, C. Katan, O. Mongin, T. Pons, J. Mertz and M. Blanchard-Desce,

Journal of Molecular Structure

A series of structurally related chromophores of different symmetry (quadrupolar, C2v, octupolar,…) and shape (rod-like, propeller-shaped, Y-shaped, dendritic,…) were investigated and compared for optimization of molecular two-photon absorption (TPA). Their design is based on the functionalization of linear or branched conjugated backbones with electron-releasing and/or electron-withdrawing peripheral groups. Their TPA spectra were determined by investigating their two-photon-excited fluorescence properties in the NIR region using pulsed excitation in the femtosecond regime. These studies provide evidence that the charge symmetry plays an important role in determining the TPA magnitude, the quadrupolar chromophore leading to the highest TPA cross-section. However, higher-order charge symmetries and branched structures provide an interesting route for improvement of the non-linear absorptivity/transparency range trade-off as well as for TPA spectral broadening in the NIR region.

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Nonlinear microscopy: new techniques and applications

Jerome Mertz,

Current Opinion in Neurobiology

Nonlinear microscopy, a general term that embraces any microscopy technique based on nonlinear optics, is further establishing itself as an important tool in neurobiology. Recent advances in labels, labeling techniques, and the use of native or genetically encoded contrast agents have bolstered the capacity of nonlinear microscopes to image the structure and function of not just single cells but of entire networks of cells. Along with novel strategies to image over exceptionally long durations and with increased depth penetration in living brains, these advances are opening new opportunities in neurobiology that were previously unavailable.

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Enhanced two-photon absorption with novel octupolar propeller-shaped fluorophores derived from triphenylamine

L. Porrès, O. Mongin, C. Katan, M. Charlot, T. Pons, J. Mertz and M. Blanchard-Desce,

Organic Letters

Novel octupolar fluorophores derived from the symmetrical functionalization of a triphenylamine core with strong acceptor peripheral groups via phenylene-ethynylene linkers have been synthesized and shown to exhibit high fluorescence quantum yields, very large TPA cross-sections in the red−NIR region, and suitable photostability.

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Two Photon Microscopy Provides Optical Sectioning

Two-photon Excited Fluorescence: Movie 1

TPEF z-stack of glomeruli in the excised olfactory bulb of an OMP-synaptopHluorin mouse. The resting GFP fluorescence indicates presynaptic regions of olfactory receptor neurons. Image width is 350μm. Sample provided by Dr. Matt Wachowiak (BU, Biology Dept.)

Second Harmonic Generation Movie

SHG z-stack from the external surface to the internal surface of an excised salamander eyeball (through sclera). Sample is unlabeled. SHG from external surface is produced by muscle fiber (scaly structures) and collagen (wavy structures). SHG from internal surface is possibly produced by axon bundles. Sample provided by Dr. Chris Passaglia (BU, BME Dept.)